Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation.     

The Hsp110 molecular chaperone stabilizes apolipoprotein B from endoplasmic reticulum-associated degradation (ERAD)

Apolipoprotein B (apoB) is the most abundant protein in low density lipoproteins and plays key roles in cholesterol homeostasis. The co-translational degradation of apoB is controlled by fatty acid levels in the endoplasmic reticulum (ER) and is mediated by the proteasome. To define the mechanism of apoB degradation, we employed a cell-free system in which proteasome-dependent degradation is recapitulated with yeast cytosol, and we developed an apoB yeast expression system. We discovered that a yeast Hsp110, Sse1p, associates with and stabilizes apoB, which contrasts with data indicating that select Hsp70s and Hsp90s facilitate apoB degradation. However, the Ssb Hsp70 chaperones have no effect on apoB turnover. To determine whether our results are relevant in mammalian cells, Hsp110 was overexpressed in hepatocytes, and enhanced apoB secretion was observed. This study indicates that chaperones within distinct complexes can play unique roles during ER-associated degradation (ERAD), establishes a role for Sse1/Hsp110 in ERAD, and identifies Hsp110 as a target to lower cholesterol.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

Nonmuscle Myosin II Is a Critical Regulator of Clathrin‐Mediated Endocytosis

Variable requirements for actin during clathrin‐mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.      

Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation

This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.     

The yeast Hsp110, Sse1p, exhibits high-affinity peptide binding

Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a K(D) of approximately 2 nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70-binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.     

Effects of grazing on plant species diversity and carbon partitioning in semiarid rangelands of northeastern China

Grasslands are one of the most widespread landscapes worldwide, covering approximately one-fifth of the world’s land surface, where grazing is a common practice. How carbon storage responds to grazing in steppes remains poorly understood. We quantified the effects of grazing on community composition and species diversity, and carbon storage in two typical grasslands of northeastern China, one in Horqin and the other one in Hulunbeier. In both grasslands, grazing did not influence plant species diversity. However, it substantially decreased aboveground carbon by 31% and 54% in Horqin and Hulunbeier, respectively. Fenced and grazing treatments showed a similar belowground carbon at both locations. The predominant carbon pool in the study grassland ecosystem was found in the upper 100 cm soil depth, from 98.2 to 99.1% of the total carbon storage. There were no significant effects of grazing on soil carbon neither in the whole profile nor in the uppermost 20 cm soil depth in the two study grasslands. Studies on the effects of varying rangeland management, such as region disparity and grazing systems, may have important consequences on species diversity and carbon partitioning, and thus on rangeland stability and ecosystem functioning.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

Overexpression of yeast Hsp110 homolog Sse1p suppresses ydj1-151 thermosensitivity and restores Hsp90-dependent activity

The Saccharomyces cerevisiae heat-shock protein (Hsp)40, Ydj1p, is involved in a variety of cellular activities that control polypeptide fate, such as folding and translocation across intracellular membranes. To elucidate the mechanism of Ydj1p action, and to identify functional partners, we screened for multicopy suppressors of the temperature-sensitive ydj1-151 mutant and identified a yeast Hsp110, SSE1. Overexpression of Sse1p also suppressed the folding defect of v-Src kinase in the ydj1-151 mutant and partially reversed the alpha-factor translocation defect. SSE1-dependent suppression of ydj1-151 thermosensitivity required the wild-type ATP-binding domain of Sse1p. However, the Sse1p mutants maintained heat-denatured firefly luciferase in a folding-competent state in vitro and restored human androgen receptor folding in sse1 mutant cells. Because the folding of both v-Src kinase and human androgen receptor in yeast requires the Hsp90 complex, these data suggest that Ydj1p and Sse1p are interacting cochaperones in the Hsp90 complex and facilitate Hsp90-dependent activity.     

Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.